Abstract

Pharmacoresistance in epilepsy is common and represents a substantial unmet need. Of the advanced therapies for focal epilepsy in preclinical development, gene delivery using viral vectors to alter neuronal or circuit excitability in epileptogenic zones is arguably the closest to clinical translation. Monogenic disorders underlying severe childhood-onset epilepsies are also candidates for such treatments, because the genetic defect may be amenable to correction, although the brain areas that need to be targeted are less clearly defined. Several gene therapies have been validated in experimental rodent models, mainly based on the delivery of DNA or RNA encoding ion channels, neurotransmitters, or receptors. Some ion channel genes, however, are too big to package into adeno-associated or lentiviral vectors, and alternative approaches to manipulate gene expression have been proposed. Current obstacles to clinical translation center on optimizing delivery to defined brain areas, determining the correct dosage, and evaluating the long-term safety and efficacy of irreversible modification of the genetic makeup of neurons.

Introduction

Epilepsy is resistant to antiseizure medication in approximately 30% of cases, and this estimate has not changed substantially despite the introduction of over 20 new drugs in the last three decades (Chen et al., 2018). Among the limitations of small molecules is that they generally do not discriminate between neurons that drive seizure generation and neurons or other non-neuronal cells supporting normal physiology. The therapeutic window for antiseizure medication is therefore often narrow. Importantly, drugs typically affect both excitatory and inhibitory neurons, and are therefore a blunt tool when compared to current mechanistic understanding of how seizures arise. Alternative therapies are urgently needed. Gene therapy, relying on the delivery of exogenous DNA or RNA, potentially overcomes the limitations of pharmacotherapy because it offers the ability to manipulate the properties of selected populations of neurons in seizure-generating circuits. It has attracted considerable attention in the last decade, motivated by two general principles. First, given the overwhelming evidence that focal (partial onset) seizures arise from a failure of inhibitory restraint, and can be triggered by stimuli that promote the firing of excitatory neurons, there is a clear path to the rational design of gene therapies that can tilt the balance of excitation and inhibition. Second, the elucidation of an expanding range of monogenic epilepsies identifies candidate defective genes which could, in principle, be replaced, supplemented, or down-regulated with gene therapy tools.

This chapter reviews recent progress in viral vectors that have allowed gene therapy to advance rapidly in neurological disorders, and the choice of promoters and transgenes that can be assembled to modify the genetic makeup of neurons targeted for treatment. Recent preclinical advances in the treatment of both focal and generalized forms of epilepsy are considered, as is the path to clinical trials.

Viral Vectors

Recent efforts in gene therapy for epilepsy build on clinical trials of gene delivery for other neurological disorders, including Parkinson disease and spinal muscular atrophy. Most studies have used viral vectors derived from adeno-associated viruses (AAVs) (Li and Samulski, 2020). These are small, single-stranded DNA parvoviruses of the Dependoparvovirus genus. Although they naturally infect humans and some apes, they are not associated with any disease. However, they cannot replicate without other “helper” viruses such as adenoviruses or herpes viruses. Recombinant AAV vectors in widespread use in the laboratory (and in clinical trials) have been modified to remove the Rep and Cap genes, which are normally translated to produce multiple distinct proteins required for replication and capsid protein synthesis. This makes AAVs replication-incompetent, and it also reduces the propensity of the viral DNA sequences to be incorporated in the host genome of infected cells (Naso et al., 2017).

AAVs can be made in cell lines by transfecting separate plasmids encoding the AAV genetic elements, the Rep and Cap genes, and the helper genes. The single-stranded linear DNA of the AAV typically encodes a promoter and the therapeutic transgene of interest, flanked up- and down-stream by inverted terminal repeats that fold back on themselves (Fig. 77–1A). For preclinical validation a reporter gene, for instance, encoding green fluorescent protein (GFP), is typically included, or else the therapeutic transgene can be tagged with an epitope for immunohistochemistry. A woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) can be included to enhance transgene expression. Following transcription, it creates a tertiary structure that enhances expression of the transgene(s), but it is not, itself, translated. After the viral particle enters a cell, the linear DNA enters the nucleus and is copied by host cell DNA polymerase complexes into double-stranded DNA. It then remains as an episome, a small circular extra-chromosomal fragment of DNA.

Figure 77–1.

Viral vectors. A. AAV, with the typical components of the single-stranded DNA (right) contained within the icosahedral capsid. A reporter protein is often included for preclinical validation. The optional WPRE sequence enhances expression. The polyadenylation (more...)

The main advantage of AAVs over many other viral vectors is that they can infect nondividing cells including neurons without eliciting cell lysis or triggering inflammation. Moreover, because neurons are postmitotic, episomes are not diluted by cell division, and so expression of transgenes can persist indefinitely. Indeed, postmortem studies from patients who participated in AAV trials for Parkinson disease have shown expression of the therapeutic transgenes over 10 years after treatment (Chu et al., 2020). There are, however, two important limitations to using AAVs. First, the packaging capacity is approximately 4.7 kilobases, which sets a limit on the size of the transgenes and regulatory elements (promoters and enhancers) that can be included. Some important epilepsy-related genes, such as those encoding voltage-gated sodium and calcium channels, exceed this capacity. Second, some people have circulating antibodies to natural strains of AAVs, and systemic delivery of therapeutic AAVs can itself trigger antibody generation. Whether such humoral immunity is relevant for intracranial delivery of AAVs, however, is unclear (Gray et al., 2013).

Far fewer clinical trials have used lentiviral vectors (lentivectors), derived from HIV-1 (Fig. 77–1B; Zufferey et al., 1998). This contains two copies of a single-stranded RNA, which contains genes encoding structural proteins, enzymes required for reverse transcription and integration into the host cell genome, and the viral envelope glycoprotein (gag, pol, and env, respectively). Modern lentivectors are typically made in cell lines co-transfected with several plasmids, which separately encode the promoter and therapeutic transgene gag and pol, and env genes, thereby minimizing the risk of recombination. The env gene is typically taken from another virus, such as vesicular stomatis, because the surface glycoprotein facilitates fusion with target cells. The lentivector particles are assembled from the proteins encoded by the viral genes and are released into the supernatant by budding. Transgene expression in cells infected by lentivectors requires the action of reverse transcriptase to make double-stranded DNA. A potential safety concern is that genetic material from the lentivector integrates into the host genome (Hacein-Bey-Abina et al., 2003), although the risk of insertional oncogenesis is very low in nondividing cells such as neurons, and can be further reduced by mutating the pol gene, which encodes integrase as well as reverse transcriptase (Wanisch and Yáñez-Muñoz, 2009). The main advantage of lentivectors is that they have a larger packaging capacity, approximately two-fold greater than that of AAVs. Lentivectors, however, are larger than AAVs, and it is more difficult to achieve widespread coverage of a brain area following intraparenchymal injection.

Viral vectors do not infect cells indiscriminately but instead exhibit natural tropism for distinct cell types. Lentivectors, for instance, have been reported to infect glial cells relatively more than AAVs (An et al., 2016). There are, however, several natural AAV serotypes, which differ with respect to the composition of the protein capsid, and the tendency of each to infect glial cells, principal neurons, and different interneuron subtypes cannot easily be extrapolated between host species. The capsids of AAV1, AAV2, AAV5, AAV8, and AAV9 have been used most extensively to target the central nervous system (CNS). Irrespective of the capsid, recombinant AAVs are typically assembled using the internal terminal repeat sequences of AAV2 and are sometimes denoted AAV2/x, where x indicates the identity of the capsid used for “pseudotyping” (e.g., AAV2/9). For the rest of this chapter, however, the recombinant AAV vectors will be denoted by their capsid identity for simplicity.

AAV9 can cross the blood–brain barrier, and this property has been further enhanced by capsid engineering (e.g., AAV-PHP.eB), allowing systemic delivery in preclinical models (Chan et al., 2017; Ravindra Kumar et al., 2020). Although this is an attractive prospect for the correction or mitigation of single gene disorders affecting the entire brain, it may be less relevant to focal epilepsy, where there is a defined target for therapy.

Other viral vectors derived from adenovirus and herpes simplex (Ingusci et al., 2019), therapies reliant on cell transplantation (Dvir et al., 2019), and nonviral methods to deliver therapeutic transgenes (Jayant et al., 2016), will not be considered here because they are currently further from clinical translation.

Promoters

Although some degree of cell type bias can be achieved by choosing an appropriate vector, far greater selectivity is usually offered through the choice of regulatory elements (promoter and enhancer sequences, for convenience referred to here as promoters) used to drive expression of the transgene. Specificity for some cell types can only be achieved at the expense of low levels of expression (Ingusci et al., 2019). Some promoters, conversely, achieve very high levels of expression, raising safety concerns if they lead to cellular stress (Xiong et al., 2019). Among powerful, relatively nonspecific, promoters are the immediate enhancer and promoter sequences from cytomegalovirus (CMV), and a sequence known as CAG assembled from fragments of the CMV early enhancer, the chicken β-actin promoter and the splice acceptor of the rabbit beta-globin gene, and the eukaryotic translation elongation factor 1α (EF-1α) promoter. Restriction of expression to neurons can be achieved with the human synapsin-1 (hSyn) promoter or the neuron-specific enolase (NSE) promoter, and further specificity for forebrain principal neurons (as opposed to interneurons) can be obtained with the promoter of Calcium/Calmodulin-dependent kinase IIα (CaMKII) (Mayford et al., 1996). Efficient expression in GABAergic interneurons has, until recently, been difficult to achieve, but some promising results have been obtained by combining regulatory sequences from transcription factors that determine interneuron development (Dimidschstein et al., 2016) or from the SCN1A gene that encodes the sodium channel Nav1.1 (Vormstein-Schneider et al., 2020).

Transgenes: Manipulation of the Excitation/Inhibition (E/I) Balance

Ultimately, the functional consequence of gene transfer depends on the choice of transgene. Many strategies to treat epilepsy have been based on the principle of promoting inhibition or decreasing excitation. This is generally in accordance with experimental evidence on the mechanisms of action of many chemoconvulsants, which interfere with inhibition or promote excitation, and of antiseizure drugs which have the opposite effects. However, in contrast to small molecules, the manipulation of neuronal or synaptic excitability can be restricted in a region and cell-type-specific manner with targeted delivery of viral vectors and choice of vector capsid and promoter. Gene therapies aiming to adjust the excitation/inhibition balance have shown promise in rat or mouse models of acquired focal (partial-onset) epilepsy, particularly in models of evoked seizures or limbic epilepsy induced by electrical kindling or chemoconvulsants.

An early study aiming to adjust the E/I balance used an AAV to express an antisense cDNA recognizing a Grin1 sequence under a CMV promoter in order to downregulate NMDA receptors. After infusion into the rat inferior colliculus, a gradual increase in threshold for electrical stimulation-induced wild running episodes was observed, arguing for an antiseizure effect (Haberman et al., 2002). Another early study used an AAV2 to overexpress the α1 GABA_A receptor subunit (encoded by Gabra1) under an α4 subunit (Gabra4) promoter, chosen because of evidence that α4 expression increases following status epilepticus (Raol et al., 2006). Injection in the rat dentate gyrus led to a subsequent attenuation of epileptogenesis evoked by a period of pilocarpine-induced status epilepticus.

Another simple strategy to decrease the excitability of principal neurons is to overexpress potassium channels (Fig. 77–2). This is a very large family, including both tetrameric voltage-gated channels, with each subunit typically containing six trans-membrane segments, and inward-rectifying channels that typically only have two transmembrane segments. To date, evidence for efficacy of potassium channel gene therapy has been reported for KCNA1 (or its rodent ortholog Kcna1), which encodes the delayed rectifier potassium channel Kv1.1 (Snowball et al., 2019; Wykes et al., 2012). This channel exists as a heterotetramer together with other Kv1 subunits as well as auxiliary beta subunits, with biophysical properties dependent on the stoichiometry. Kv1.1 (or to be precise Kv1.1-containing) channels are principally expressed in axons and presynaptic boutons of a wide range of neurons in the CNS and periphery, where they affect action potential initiation threshold as well as presynaptic spike width and, consequently, presynaptic calcium influx and neurotransmitter release. In vitro overexpression experiments have shown that neuronal excitability and neurotransmitter release are both attenuated by Kv1.1 overexpression (Heeroma et al., 2009), and a reduction in firing rate has also been confirmed in ex vivo tissue following lentiviral overexpression in the motor cortex (Wykes et al., 2012). These effects were graded, however, and neuronal or synaptic silencing was not observed. On this basis, Kv1.1 overexpression has been trialed in several rat models, using either the wild-type human sequence or a codon-optimized version with an additional mutation that mimics the effect of posttranscriptional editing, with a subtle effect on inactivation kinetics (Snowball et al., 2019). It has also been delivered with several different vector and promoter combinations, including a nonintegrating lentivector and AAV9, and the CMV and human CaMKII promoter (Snowball et al., 2019; Wykes et al., 2012). Ex vivo immunofluorescence showed a strong bias to expression in pyramidal neurons over interneurons or glial cells even when using the CMV promoter (Wykes et al., 2012). These studies showed an effect both on the evolution of seizures after an epileptogenic insult (Wykes et al., 2012) but also, closer to a clinical trial design, on established epilepsy, with decreases in seizure frequency and (in the case of a temporal lobe epilepsy model) seizure duration (Snowball et al., 2019; Wykes et al., 2012). When injected in the motor cortex of control rats, no effect was seen on a test of sensorimotor coordination (Wykes et al., 2012), arguing that it may be well tolerated in the clinic.

Figure 77–2.

Representative transgenes used in preclinical gene therapies for focal epilepsy, with putative mechanisms of action. Kv1.1 overexpression reduces neuronal excitability and shortens action potentials, thereby reducing neurotransmitter release. eGluCl (more...)

Neither NMDA receptor downregulation nor potassium channel overexpression is, by design, aimed to differentiate between normal and pathological circuit activity. Overexpression of inhibitory neuropeptides has the theoretical advantage that the release of such transmitters generally requires intense activity such as might occur during seizures. Several neuropeptides, including galanin, neuroptide Y, somatostatin, ghrelin, and dynorphin, also have antiseizure effects when administered in vivo (Burtscher and Schwarzer, 2017; Kovac and Walker, 2013). These considerations have motivated several attempts to use neuropeptides as transgenes to treat rodent models of temporal lobe epilepsy. The earliest such studies used galanin, fused to a fibronectin secretory signal sequence, placed under a CMV promoter and packaged in an AAV (Haberman et al., 2003). After injection into the inferior colliculus, there was an increase in the threshold for electrical stimulation-induced wild running. A similar effect was observed when galanin was placed under a doxycycline-off promoter, a design that potentially allows the treatment to be regulated by administration of the antibiotic doxycycline. Furthermore, the gene therapy delivered to the hippocampal formation attenuated kainic acid–induced hilar cell death, implying a neuroprotective effect. An antiseizure effect was also reported when galanin was overexpressed in the pyriform cortex (McCown, 2006). Subsequent studies reported that galanin, expressed using an NSE promoter, attenuated seizures in hippocampal epilepsy models, but without affecting epileptogenesis or kindling development (Kanter-Schlifke et al., 2007; Lin et al., 2003). Somatostatin, expressed under a CAG promoter in an AAV5, has also been reported to attenuate seizures in a kindling model (Natarajan et al., 2017; Zafar et al., 2012).

Of the neuropeptides, NPY has arguably received the most attention as a therapeutic transgene (Fig. 77–2). It is mainly expressed in interneurons and acts via G protein–coupled receptors (Y1 through Y5), several of which are expressed presynaptically by glutamatergic neurons (Cattaneo et al., 2021). Several studies have shown an attenuation of acute chemoconvulsant-evoked seizures following overexpression in the pyriform cortex (Foti et al., 2007) or of seizures in temporal lobe epilepsy models (Noe et al., 2010; Richichi et al., 2004; Sørensen et al., 2009). Evidence for an antiseizure effect after epilepsy has developed has been reported for pre-pro-NPY expressed under a CAG-like promoter in a chimeric AAV1-2 vector (Noè et al., 2008). NPY gene therapy, using an NSE promoter in an AAV1, has also been shown to suppress seizures in the Genetic Absence Epileptic Rats from Strasbourg (GAERS) model of genetic generalized epilepsy following expression in either the ventrolateral thalamus or the somatosensory cortex (Powell et al., 2018).

A potential limitation of NPY overexpression is that the neuropeptide acts on several receptors, at least one of which, Y1, has been reported to have pro-epileptic actions (Gariboldi et al., 1998). This has prompted attempts to improve the antiepileptic effect of NPY gene therapy by simultaneously overexpressing Y2 (Woldbye et al., 2010) or Y5 (Gøtzsche et al., 2012). These studies used coinjection of two similar AAVs with NSE promoters driving the expression of NPY or its receptor. In a subsequent study, human pre-pro-NPY and Y2 receptor genes, linked by an internal ribosome entry site (IRES) sequence, were packaged together with a CAG promoter, and shown to reduce seizures in a chronic epilepsy model (Melin et al., 2019). This strategy is being advanced to clinical trials by Combigene.

Regulated Gene Therapy

A potential limitation of constitutive manipulation of the E/I balance is that, because gene therapy is invasive and irreversible, it is difficult to ensure that the dosage is correct. Underdosage (whether expressed as viral copy numbers per neuron or too sparse or restricted an area of treated neurons) may fail to achieve an antiepileptic effect. Overinhibition, conversely, may render a brain area relatively unexcitable, with potentially deleterious effects on essential brain functions. This consideration underlines the importance not only of quantifying the effect of gene therapy on seizure frequency and/or severity in preclinical models but also of looking for off target effects. Because many gene therapies have been tested in models of temporal lobe epilepsy, with treatment targeted to the hippocampal formation, in practice this means measuring learning and memory. In the case of neocortical epilepsy models, sensory or motor function should be examined using tests that are sensitive to lesions of the corresponding brain areas.

Although several such studies have shown that seizure attenuation can be achieved without impairing memory or motor coordination, it is not trivial to extrapolate from a rodent model to the human brain. The concerns surrounding safety and dosage have prompted a search for gene therapy strategies that can be regulated by an external factor. Both optogenetics and chemogenetics are potential solutions to this challenge, and both have been shown to be effective in rodent models. In the case of optogenetics, both inhibitory opsins targeted to excitatory neurons and excitatory opsins targeted to inhibitory neurons have been shown to suppress seizures on demand, by delivering light to the treated brain area (Hristova et al., 2021; Krook-Magnuson et al., 2013; Paz et al., 2013; Takeuchi et al., 2021; Wicker and Forcelli, 2021; Wykes et al., 2012). However, the challenge to clinical translation of optogenetics for epilepsy is not trivial. The opsins themselves are not mammalian proteins, with the potential to trigger immune attack, although this has not prevented a clinical trial in retinitis pigmentosa (Sahel et al., 2021). In addition, optogenetics requires implantable devices to deliver light to the brain, which represents a risk of infection and equipment failure. Nevertheless, optogenetics offers the prospect of quasi-instantaneous closed-loop manipulation of excitability upon detection of epileptiform activity (Hristova et al., 2021; Krook-Magnuson et al., 2013; Paz et al., 2013), with greater cell type specificity and potentially less tissue damage than electrical deep brain stimulation, which has already been approved for use in the clinic (Morrell and RNS System in Epilepsy Study Group, 2011).

Chemogenetics is arguably closer to clinical translation than optogenetics because it avoids the need for invasive light delivery devices (Walker and Kullmann, 2020). It, however, acts on a slower timescale, mainly determined by the pharmacokinetics of the ligand. Several studies have demonstrated efficacy of chemogenetics using muscarinic DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) (Cǎlin et al., 2018; Desloovere et al., 2019; Goossens et al., 2021; Kätzel et al., 2014; Wicker and Forcelli, 2016). hM4Di is a versatile inhibitory DREADD derived from the human M4 Gi-coupled receptor, with two amino acid substitutions rendering it insensitive to its endogenous agonist acetylcholine, but allowing activation by exogenous drugs such as clozapine, which is used in the treatment of psychosis, and its otherwise inactive metabolite clozapine-N-oxide (CNO). In an early study, reversible seizure suppression was achieved following AAV5-mediated expression of hM4Di under a CaMKII promoter in principal neurons in the neocortex upon intraperitoneal administration of CNO (Kätzel et al., 2014). It subsequently emerged that CNO does not penetrate the CNS efficiently, and most likely acts through back-conversion to clozapine, which crosses the blood–brain barrier (Gomez et al., 2017; Manvich et al., 2018). Clozapine itself has been used to achieve seizure suppression in a temporal lobe epilepsy model where hM4Di was expressed using an AAV7 (Desloovere et al., 2019). The antiseizure effect persisted with repeated dosing for several days. For clinical translation, however, clozapine is a suboptimal ligand because it is associated with a substantial risk of agranulocytosis, and has even been reported to be pro-convulsant (Ruffmann et al., 2006). Although other powerful and selective agonists have been developed, they are not approved for clinical use. Another antipsychotic drug in widespread use, olanzapine, could, however, be repurposed as an activating ligand (Weston et al., 2019) and is effective in suppressing seizures in rats expressing hM4Di in the hippocampus (Goossens et al., 2021) (Fig. 77–2).

Chemogenetic treatment has also been reported following thalamic expression of hM4Di under an hSyn promoter with an AAV8 in an amygdala kindling model (Wicker and Forcelli, 2016). A pro-excitatory counterpart of hM4Di, hM3Dq, has also been used to suppress seizures following expression in interneurons (Wang et al., 2018).

Another group of chemogenetic actuators act as ligand-gated ion channels (LGICs), although have not been studied extensively as antiseizure tools (Lieb et al., 2019). Available inhibitory LGIC chemogenetic actuators inhibit neurons by opening a chloride conductance. This has a potential limitation, shared with antiseizure drugs acting on GABA_A receptors, that much of their inhibitory effect may be lost if the trans-membrane driving force for chloride ions collapses, as has been reported in experimental models of epilepsy (Magloire et al., 2019, 2018). Nevertheless, a chloride-permeable LGIC chemogenetic actuator that dispenses with the need for an exogenous ligand has been used to treat experimental epilepsy. This is a glutamate-gated chloride channel from C. elegans that has been mutated to increase its sensitivity to glutamate (eGluCl) (Lieb et al., 2018). Extracellular glutamate elevation, as occurs in seizure foci (During and Spencer, 1993), activates the channel and inhibits neurons in closed loop. It remains to be determined to which extent the antiseizure effect of eGluCl relies on a hyperpolarizing effect of opening the chloride conductance as opposed to shunting of excitatory currents (Fig. 77–2).

Upregulation of Endogenous Gene Expression

All of the above antiepileptic strategies (with the exception of targeting Grin1 with an antisense cDNA; Haberman et al., 2002) rely on the expression of exogenous genes. An alternative solution is to up- or down-regulate endogenous gene expression. This can be achieved by targeting endogenous microRNAs (Morris et al., 2021), but it will not be considered further here because it is not a gene therapy as such. As for manipulating transcription, some emerging strategies rely on CRISPR (clustered regularly interspaced short palindromic repeats) technology. CRISPR activation and CRISPR interference make use of a nuclease-deficient Cas9 protein fused to transcriptional activator or repressor domains respectively, together with a guide RNA designed to recognize regulatory sequences of the desired gene (Dominguez et al., 2016). A theoretical advantage of these approaches is that post-transcriptional processes such as splicing are unaffected. In principle, more than one gene could be targeted simultaneously by using several guide RNAs. To date, upregulation of endogenous genes using CRISPR activation has only been tested with a single-guide RNA at a time. A murine model of temporal lobe epilepsy responded to CRISPR activation of Kcna1 (Colasante et al., 2020b), and a model of Dravet syndrome responded to upregulation of Scn1a (Colasante et al., 2020a; Yamagata et al., 2020; see below). The dCas9 and guide RNA were delivered using two viruses (Colasante et al., 2020b, 2020a), but compact alternatives to Cas9 are emerging (Pausch et al., 2020), potentially allowing the entire CRISPR machinery to be contained within a single therapeutic AAV.

The study describing CRISPR activation of Kcna1 demonstrated not only a reduction in seizure frequency but also rescue of a cognitive comorbidity and partial correction of transcriptional dysregulation (Colasante et al., 2020b). Chemogenetic inhibition of dentate granule cells has also been shown to ameliorate performance in a hippocampus-dependent memory task (Kahn et al., 2019). These observations raise the tantalizing prospect that gene therapy could go beyond mere treatment of symptoms (i.e., seizures) and potentially reverse aspects of epileptogenesis.

Neurotrophic Factors

A potential limitation of the strategies summarized above is that they do not address other features of acquired epilepsy, such as the cell death and aberrant synaptic connections that frequently accompany epileptogenesis. A promising approach to mitigate these phenomena is to manipulate neurotrophin signaling. An early study using an AAV to overexpress glial-derived neurotrophic factor (GDNF) reported an antiepileptic effect, although it did not examine neurodegeneration (Kanter-Schlifke et al., 2007). Another study used a modified herpes simplex (HS) vector to overexpress both fibroblast-derived growth factor-2 (FGF2) and brain-derived neurotrophic factor (BDNF) in the hippocampal formation (Paradiso et al., 2009). HS-derived vectors have a much larger packaging capacity than AAVs or lentivectors, and in addition, they can be retrogradely transported and lead to transient expression, potentially useful properties to interfere with epileptogenesis following a brain insult. When injected following a period of status epilepticus, this combined FGF2 and BDNF gene therapy led to a reduction in cell death, inflammation, mossy fiber sprouting, and epileptogenesis (Bovolenta et al., 2010; Paradiso et al., 2011, 2009).

Monogenic Epilepsies

Recent years have seen rapid progress in gene therapy for developmental and epileptic encephalopathies (DEEs), mainly driven by the identification of monogenic causes. It is reasonable to hypothesize that correcting the genetic defect has an antiseizure effect and may also arrest disease progression or even reverse some of the intellectual disability or other manifestations of these disorders. The remarkable success of genetic treatments for spinal muscular atrophy (Mendell et al., 2017) has led to considerable optimism that analogous therapies for DEEs will be made available. However, there are several potential obstacles. First, the mechanisms by which seizures and other features of DEEs arise are incompletely understood. Indeed, some mouse models only incompletely recapitulate the disorders. Second, widespread and uniform dosage of therapeutic transgenes is difficult to achieve using viral vectors. Overdosage at the level of individual neurons may, for instance, be deleterious. Third, the nature of the genetic defect may not lend itself to simple supplementation with an exogenous transgene. For instance, some disorders probably arise as a gain of abnormal function or through a dominant negative effect (Kang and Macdonald, 2016). A final consideration is that the mutations may have an irreversible effect on brain development, and the optimal window for treatment may be difficult to identify. Nevertheless, there have been some promising advances in the treatment of mouse models of some DEEs, and some clinical trials are imminent.

Of the DEEs, Dravet syndrome has probably attracted the most attention, prompted by the recognition that it is caused in over 70% of cases by heterozygous loss-of-function mutations of the SCN1A gene, encoding Nav1.1 (Claes et al., 2001). There is, moreover, strong evidence that GABAergic interneurons are especially intolerant of haploinsufficiency (Yu et al., 2006). This prompts the need to increase SCN1A function specifically in these cells. Further complicating the challenge is that the coding sequence (approximately 6 kb) exceeds the packaging capacity of AAVs, and the limited spread of lentiviral vectors makes it difficult to envisage efficient distribution throughout the brain. Several alternative approaches have, however, been tested in mouse models. First, CRISPR activation using a dead Cas9, analogous to the treatment of temporal lobe epilepsy with upregulation of Kcna1 transcription (Colasante et al., 2020b), has been used by two groups (Colasante et al., 2020a; Yamagata et al., 2020). This approach relies on the identification of guide RNAs that are able to selectively increase Scn1a transcription. Assuming that increased expression of the mutant allele has no deleterious consequences, a simple two-fold increase in transcription of the wild-type allele should, in principle, be sufficient to achieve a correction of the genetic defect. Furthermore, as mentioned above, CRISPR activation should not affect splicing and trafficking, thus preserving normal ion channel biogenesis. In one of the cited studies, only the guide RNA was delivered with an AAV, while the remainder of the CRISPR machinery was expressed constitutively in GABAergic interneurons by crossing a mouse line with a floxed dCas9 fused to transcriptional enhancers with a Vgat-Cre driver line further crossed into a haploinsufficient Scn1a line (Yamagata et al., 2020). In the other study, arguably closer to clinical translation, the two halves of the CRISPR machinery were divided into two AAVs, one expressing dCas9 fused to transcriptional activator domains, under the control of the reverse tetracycline-controlled transactivator (rtTA)-responsive element (TRE), and the other expressing the guide RNA and the rtTA under the mDlx5/6 forebrain interneuron-specific promoter (Colasante et al., 2020a). In this way, in the presence of the tetracycline analog doxycycline, transcriptional enhancers were recruited to the Scn1a promoter exclusively in interneurons. However, only approximately 20% of forebrain interneurons were co-transduced, and the treatment was achieved with early postnatal intracerebroventricular AAV injection followed by continuous doxycycline administration, calling for more extensive exploration of the optimal dosage and time of intervention. Nevertheless, both approaches led to an attenuation of hyperthermia-induced seizures (Colasante et al., 2020a; Yamagata et al., 2020), and the mouse transgenic strategy also mitigated some cognitive deficits (Yamagata et al., 2020).

A further study took an alternative approach to treat a mouse model of Dravet syndrome, based on overexpression of the auxiliary sodium channel subunit Navβ1. A dominantly inherited mutation of the encoding gene SCN1B was the first elucidated epileptic sodium channelopathy, identified in a large family with generalized epilepsy with febrile seizures plus (GEFS+) (Wallace et al., 1998). Neonatal intracerebroventricular injection of AAV9 with Scn1b under a truncated mouse Gad1 promoter led to a reduction in mortality and hyperthermia-induced seizures, but with some unexpected sex differences (Niibori et al., 2020). It remains to be determined whether the therapeutic effect is accompanied by an alteration of Nav1.1-mediated sodium current density in interneurons or other cells.

Among other strategies to treat Dravet syndrome, one relies on antisense oligonucleotides to suppress specifically a naturally nonproductive splice variant of the Scn1a gene, with the result that translation of the wild-type allele is increased. This approach, known as targeted augmentation of nuclear gene output (TANGO), is strictly speaking a form of RNA rather than gene therapy (Han et al., 2020), and it is being developed by Stoke Therapeutics with an open-label clinical trial under way at the time of writing. Another RNA therapy is based on the discovery of natural antisense transcripts that limit Scn1a mRNA availability. Antisense oligonucleotides targeting such transcripts (“AntagoNATs”) (Hsiao et al., 2016) are being developed by OPKO Health. Antisense therapies are a more obvious treatment for gain-of-function mutations, and they have been tested in a mouse model of Scn8a-associated epileptic encephalopathy (Lenk et al., 2020).

As for other gene therapies for Dravet syndrome, only very preliminary data have been reported in abstract form for the ability of AAV-based targeting of endogenous regulatory elements to upregulate Scn1a transcription specifically in interneurons (Young et al., 2019). This is being developed by Encoded Therapeutics.

Rett syndrome has a similar incidence to Dravet syndrome, although almost exclusively affects girls, and it is mainly caused by heterozygous mutations of the X-linked MECP2 gene (Amir et al., 1999). These mutations are generally loss-of-function, and because of X-inactivation, affected individuals are effectively mosaic for normal versus mutant alleles. Seizures are a major feature of Rett syndrome, and they can be rescued in mouse models by delayed reactivation, achieved by switching on a “stop-flox” Mecp2 allele in mice coexpressing an estrogen receptor/Cre-recombinase (Lang et al., 2014). This underlines the potential of gene therapy even after symptoms have begun to manifest. Indeed, AAV9-mediated Mecp2 gene delivery has been reported to ameliorate some behavioral abnormalities in mouse models of Rett syndrome, although only with preliminary data on seizures (Bassuk, n.d.; Garg et al., 2013). However, a major challenge to gene therapy strategies aimed at upregulation or replacement of MECP2 is that another neurological syndrome is associated with gene duplication (Ramocki, 2010). A strategy that incorporates a microRNA to achieve feedback regulation of Mecp2 dosage to mitigate this risk has recently been reported (Sinnett et al., 2021).

An ambitious goal for treatment of monogenic disorders would be to correct the genomic defect permanently. Until recently, the application of CRISPR-Cas9 technology to achieve genome editing in the CNS (as opposed to transcriptional modulation) was limited by the inefficiency of homology-directed repair mechanisms in nondividing cells. However, a promising approach, homology-independent targeted integration (HITI) potentially overcomes this barrier and has been used to achieve knock-in of entire exons and to treat a model of retinitis pigmentosa (Suzuki et al., 2016). The potential of this approach, and other methods to edit the genome, to treat DEEs has recently been reviewed (Turner et al., 2021).

Clinical Translation

Clinical trials of gene (as opposed to antisense oligonucleotide) therapy for refractory epilepsy have not, at the time of writing, started to recruit patients. For monogenic epilepsies a substantial challenge is to determine how and where to deliver the viral vector to achieve optimal coverage of neurons responsible for seizures and other features of the disorders. This question is not fully resolved in the case of focal epilepsies, too: there is evidence for efficacy of optogenetic and chemogenetic therapy targeted to the thalamus (Wicker and Forcelli, 2021, 2016), medial septum (Hristova et al., 2021), and even cerebellum (Krook-Magnuson et al., 2014) in rodent temporal lobe epilepsy models. Nevertheless, for the purpose of first in human trials, gene therapy targeted to regions that can be resected offers distinct advantages. First, it provides an option to remove the transduced region in the event of serious adverse events. And second, if the treatment is ineffective patients could proceed to definitive epilepsy surgery, providing tissue for quantification of the therapeutic transgene or protein, expressed in terms of the spatial extent, cell type and proportion of neurons successfully transduced, and possibly even number of episomal DNA copies per cell. These advantages are not shared with experimental or established gene therapies used to treat inherited or neurodegenerative diseases. For DREADD-based chemogenetic epilepsy therapy, it may even be possible to obtain post hoc evidence of target engagement without resecting tissue, because ¹¹C-clozapine can be used as a PET ligand to detect expression noninvasively (Nagai et al., 2016).

Nevertheless, there are no doubt lessons to be learned from clinical trials of gene therapy for such disorders as Parkinson disease. In particular, considerable advances have been made in optimizing the design of injection cannulae, and titre, volume, and speed of infusion of viral vector suspensions. One interesting approach is to coinfuse a gadolinium-based contrast agent and to obtain magnetic resonance imaging (MRI) to obtain quasi-real-time data on the extent of spread of the therapy (Lonser et al., 2020).

Conclusions

Gene therapy for epilepsy is progressing across several disease subtypes. It builds on advances in understanding the mechanisms of seizure generation, in both acquired and genetic epilepsies, and a clinical trial is due to start recruiting imminently (University College, London, 2020).

Disclosure Statement

References

1. Amir, R.E., Van den Veyver, I.B., Wan, M., Tran, C.Q., Francke, U., Zoghbi, H.Y., 1999. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.  Nat Genet 23, 185–188. https://doi​.org/10.1038/13810 [PubMed: 10508514]
2. An, H., Cho, D.-W., Lee, S.E., Yang, Y.-S., Lee, S.-C.H. and C.J., 2016. Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey.  Experimental Neurobiology 25, 48–54. https://doi​.org/10.5607/en.2016.25.1.48 [PMC free article: PMC4766114] [PubMed: 26924933]
3. Bassuk, A.G., n.d. Gene therapy for Rett syndrome. Genes, Brain and Behavior, e12754. https://doi​.org/10.1111/gbb.12754 [PMC free article: PMC9744469] [PubMed: 34053173]
4. Bovolenta, R., Zucchini, S., Paradiso, B., Rodi, D., Merigo, F., Navarro Mora, G., Osculati, F., Berto, E., Marconi, P., Marzola, A., Fabene, P.F., Simonato, M., 2010. Hippocampal FGF-2 and BDNF overexpression attenuates epileptogenesis-associated neuroinflammation and reduces spontaneous recurrent seizures.  J Neuroinflammation 7, 81. https://doi​.org/10.1186/1742-2094-7-81 [PMC free article: PMC2993685] [PubMed: 21087489]
5. Burtscher, J., Schwarzer, C., 2017. The Opioid System in Temporal Lobe Epilepsy: Functional Role and Therapeutic Potential.  Front. Mol. Neurosci. 0. https://doi​.org/10.3389/fnmol.2017.00245 [PMC free article: PMC5545604] [PubMed: 28824375]
6. Cǎlin, A., Stancu, M., Zagrean, A.-M., Jefferys, J.G.R., Ilie, A.S., Akerman, C.J., 2018. Chemogenetic Recruitment of Specific Interneurons Suppresses Seizure Activity.  Front Cell Neurosci 12, 293. https://doi​.org/10.3389/fncel.2018.00293 [PMC free article: PMC6134067] [PubMed: 30233328]
7. Cattaneo, S., Verlengia, G., Marino, P., Simonato, M., Bettegazzi, B., 2021. NPY and Gene Therapy for Epilepsy: How, When,… and Y.  Front. Mol. Neurosci. 0. https://doi​.org/10.3389/fnmol​.2020.608001 [PMC free article: PMC7862707] [PubMed: 33551745]
8. Chan, K.Y., Jang, M.J., Yoo, B.B., Greenbaum, A., Ravi, N., Wu, W.-L., Sánchez-Guardado, L., Lois, C., Mazmanian, S.K., Deverman, B.E., Gradinaru, V., 2017. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.  Nat Neurosci 20, 1172–1179. https://doi​.org/10.1038/nn.4593 [PMC free article: PMC5529245] [PubMed: 28671695]
9. Chen, Z., Brodie, M.J., Liew, D., Kwan, P., 2018. Treatment Outcomes in Patients With Newly Diagnosed Epilepsy Treated With Established and New Antiepileptic Drugs: A 30-Year Longitudinal Cohort Study.  JAMA Neurol 75, 279–286. https://doi​.org/10.1001/jamaneurol​.2017.3949 [PMC free article: PMC5885858] [PubMed: 29279892]
10. Chu, Y., Bartus, R.T., Manfredsson, F.P., Olanow, C.W., Kordower, J.H., 2020. Long-term post-mortem studies following neurturin gene therapy in patients with advanced Parkinson’s disease.  Brain 143, 960–975. https://doi​.org/10.1093/brain/awaa020 [PMC free article: PMC7089653] [PubMed: 32203581]
11. Claes, L., Del-Favero, J., Ceulemans, B., Lagae, L., Van Broeckhoven, C., De Jonghe, P., 2001. De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy.  Am. J. Hum. Genet 68, 1327–1332. https://doi​.org/10.1086/320609 [PMC free article: PMC1226119] [PubMed: 11359211]
12. Colasante, G., Lignani, G., Brusco, S., Di Berardino, C., Carpenter, J., Giannelli, S., Valassina, N., Bido, S., Ricci, R., Castoldi, V., Marenna, S., Church, T., Massimino, L., Morabito, G., Benfenati, F., Schorge, S., Leocani, L., Kullmann, D.M., Broccoli, V., 2020a. dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice. Mol. Ther. 28, 235–253. https://doi​.org/10.1016/j​.ymthe.2019.08.018 [PMC free article: PMC6952031] [PubMed: 31607539]
13. Colasante, G., Qiu, Y., Massimino, L., Di Berardino, C., Cornford, J.H., Snowball, A., Weston, M., Jones, S.P., Giannelli, S., Lieb, A., Schorge, S., Kullmann, D.M., Broccoli, V., Lignani, G., 2020b. In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy.  Brain 143, 891–905. https://doi​.org/10.1093/brain/awaa045 [PMC free article: PMC7089667] [PubMed: 32129831]
14. Desloovere, J., Boon, P., Larsen, L.E., Merckx, C., Goossens, M.-G., Van den Haute, C., Baekelandt, V., De Bundel, D., Carrette, E., Delbeke, J., Meurs, A., Vonck, K., Wadman, W., Raedt, R., 2019. Long-term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy. Epilepsia. https://doi​.org/10.1111/epi.16368 [PubMed: 31608439]
15. Dimidschstein, J., Chen, Q., Tremblay, R., Rogers, S.L., Saldi, G.-A., Guo, L., Xu, Q., Liu, R., Lu, C., Chu, J., Grimley, J.S., Krostag, A.-R., Kaykas, A., Avery, M.C., Rashid, M.S., Baek, M., Jacob, A.L., Smith, G.B., Wilson, D.E., Kosche, G., Kruglikov, I., Rusielewicz, T., Kotak, V.C., Mowery, T.M., Anderson, S.A., Callaway, E.M., Dasen, J.S., Fitzpatrick, D., Fossati, V., Long, M.A., Noggle, S., Reynolds, J.H., Sanes, D.H., Rudy, B., Feng, G., Fishell, G., 2016. A viral strategy for targeting and manipulating interneurons across vertebrate species.  Nat Neurosci 19, 1743–1749. https://doi​.org/10.1038/nn.4430 [PMC free article: PMC5348112] [PubMed: 27798629]
16. Dominguez, A.A., Lim, W.A., Qi, L.S., 2016. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation.  Nat Rev Mol Cell Biol 17, 5–15. https://doi​.org/10.1038/nrm.2015.2 [PMC free article: PMC4922510] [PubMed: 26670017]
17. During, M.J., Spencer, D.D., 1993. Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.  Lancet 341, 1607–1610. [PubMed: 8099987]
18. Dvir, N., Javaid, M.S., Jones, N.C., Powell, K.L., Kwan, P., O’Brien, T.J., Antonic-Baker, A., 2019. The effects of cell therapy on seizures in animal models of epilepsy: protocol for systematic review and meta-analysis of preclinical studies.  Systematic Reviews 8, 255. https://doi​.org/10.1186​/s13643-019-1169-3 [PMC free article: PMC6824117] [PubMed: 31675988]
19. Foti, S., Haberman, R.P., Samulski, R.J., McCown, T.J., 2007. Adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13–36 suppresses seizure activity in vivo.  Gene Ther 14, 1534–1536. https://doi​.org/10.1038/sj.gt.3303013 [PMC free article: PMC3557464] [PubMed: 17713567]
20. Garg, S.K., Lioy, D.T., Cheval, H., McGann, J.C., Bissonnette, J.M., Murtha, M.J., Foust, K.D., Kaspar, B.K., Bird, A., Mandel, G., 2013. Systemic Delivery of MeCP2 Rescues Behavioral and Cellular Deficits in Female Mouse Models of Rett Syndrome.  J. Neurosci. 33, 13612–13620. https://doi​.org/10.1523/JNEUROSCI​.1854-13.2013 [PMC free article: PMC3755711] [PubMed: 23966684]
21. Gariboldi, M., Conti, M., Cavaleri, D., Samanin, R., Vezzani, A., 1998. Anticonvulsant properties of BIBP3226, a non-peptide selective antagonist at neuropeptide Y Y1 receptors.  Eur J Neurosci 10, 757–759. https://doi​.org/10.1046/j​.1460-9568.1998.00061.x [PubMed: 9749738]
22. Gomez, J.L., Bonaventura, J., Lesniak, W., Mathews, W.B., Sysa-Shah, P., Rodriguez, L.A., Ellis, R.J., Richie, C.T., Harvey, B.K., Dannals, R.F., Pomper, M.G., Bonci, A., Michaelides, M., 2017. Chemogenetics revealed: DREADD occupancy and activation via converted clozapine.  Science 357, 503–507. https://doi​.org/10.1126/science.aan2475 [PMC free article: PMC7309169] [PubMed: 28774929]
23. Goossens, M.-G., Boon, P., Wadman, W., Van den Haute, C., Baekelandt, V., Verstraete, A.G., Vonck, K., Larsen, L.E., Sprengers, M., Carrette, E., Desloovere, J., Meurs, A., Delbeke, J., Vanhove, C., Raedt, R., 2021. Long-term chemogenetic suppression of seizures in a multifocal rat model of temporal lobe epilepsy.  Epilepsia 62, 659–670. https://doi​.org/10.1111/epi.16840 [PubMed: 33570167]
24. Gøtzsche, C.R., Nikitidou, L., Sørensen, A.T., Olesen, M.V., Sørensen, G., Christiansen, S.H.O., Ängehagen, M., Woldbye, D.P.D., Kokaia, M., 2012. Combined gene overexpression of neuropeptide Y and its receptor Y5 in the hippocampus suppresses seizures.  Neurobiol Dis 45, 288–296. https://doi​.org/10.1016/j​.nbd.2011.08.012 [PubMed: 21884793]
25. Gray, S.J., Nagabhushan Kalburgi, S., McCown, T.J., Jude Samulski, R., 2013. Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates.  Gene Ther 20, 450–459. https://doi​.org/10.1038/gt.2012.101 [PMC free article: PMC3618620] [PubMed: 23303281]
26. Haberman, R.P., Criswell, H.E., Snowdy, S., Ming, Z., Breese, G.R., Samulski, R.J., McCown, T.J., 2002. Therapeutic Liabilities of in Vivo Viral Vector Tropism: Adeno-Associated Virus Vectors, NMDAR1 Antisense, and Focal Seizure Sensitivity.  Molecular Therapy 6, 495–500. https://doi​.org/10.1006/mthe.2002.0701 [PMC free article: PMC3213639] [PubMed: 12377191]
27. Haberman, R.P., Samulski, R.J., McCown, T.J., 2003. Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion.  Nat Med 9, 1076–1080. https://doi​.org/10.1038/nm901 [PubMed: 12858168]
28. Hacein-Bey-Abina, S., von Kalle, C., Schmidt, M., Le Deist, F., Wulffraat, N., McIntyre, E., Radford, I., Villeval, J.-L., Fraser, C.C., Cavazzana-Calvo, M., Fischer, A., 2003. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency.  N Engl J Med 348, 255–256. https://doi​.org/10.1056​/NEJM200301163480314 [PubMed: 12529469]
29. Han, Z., Chen, C., Christiansen, A., Ji, S., Lin, Q., Anumonwo, C., Liu, C., Leiser, S.C., Meena, Aznarez, I., Liau, G., Isom, L.L., 2020. Antisense oligonucleotides increase Scn1a expression and reduce seizures and SUDEP incidence in a mouse model of Dravet syndrome. Science Translational Medicine 12. https://doi​.org/10.1126/scitranslmed​.aaz6100 [PubMed: 32848094]
30. Heeroma, J.H., Henneberger, C., Rajakulendran, S., Hanna, M.G., Schorge, S., Kullmann, D.M., 2009. Episodic ataxia type 1 mutations differentially affect neuronal excitability and transmitter release.  Dis Model Mech 2, 612–619. https://doi​.org/10.1242/dmm.003582 [PMC free article: PMC2773728] [PubMed: 19779067]
31. Hristova, K., Martinez-Gonzalez, C., Watson, T.C., Codadu, N.K., Hashemi, K., Kind, P.C., Nolan, M.F., Gonzalez-Sulser, A., 2021. Medial septal GABAergic neurons reduce seizure duration upon optogenetic closed-loop stimulation.  Brain 144, 1576–1589. https://doi​.org/10.1093/brain/awab042 [PMC free article: PMC8219369] [PubMed: 33769452]
32. Hsiao, J., Yuan, T.Y., Tsai, M.S., Lu, C.Y., Lin, Y.C., Lee, M.L., Lin, S.W., Chang, F.C., Liu Pimentel, H., Olive, C., Coito, C., Shen, G., Young, M., Thorne, T., Lawrence, M., Magistri, M., Faghihi, M.A., Khorkova, O., Wahlestedt, C., 2016. Upregulation of Haploinsufficient Gene Expression in the Brain by Targeting a Long Non-coding RNA Improves Seizure Phenotype in a Model of Dravet Syndrome.  EBioMedicine 9, 257–277. https://doi​.org/10.1016/j​.ebiom.2016.05.011 [PMC free article: PMC4972487] [PubMed: 27333023]
33. Ingusci, S., Verlengia, G., Soukupova, M., Zucchini, S., Simonato, M., 2019. Gene Therapy Tools for Brain Diseases.  Front Pharmacol 10, 724. https://doi​.org/10.3389/fphar.2019.00724 [PMC free article: PMC6613496] [PubMed: 31312139]
34. Jayant, R.D., Sosa, D., Kaushik, A., Atluri, V., Vashist, A., Tomitaka, A., Nair, M., 2016. Current status of non-viral gene therapy for CNS disorders.  Expert Opin Drug Deliv 13, 1433–1445. https://doi​.org/10.1080/17425247​.2016.1188802 [PMC free article: PMC5480312] [PubMed: 27249310]
35. Kahn, J.B., Port, R.G., Yue, C., Takano, H., Coulter, D.A., 2019. Circuit-based interventions in the dentate gyrus rescue epilepsy-associated cognitive dysfunction.  Brain 142, 2705–2721. https://doi​.org/10.1093/brain/awz209 [PMC free article: PMC6736326] [PubMed: 31363737]
36. Kang, J.-Q., Macdonald, R.L., 2016. Molecular Pathogenic Basis for GABRG2 Mutations Associated With a Spectrum of Epilepsy Syndromes, From Generalized Absence Epilepsy to Dravet Syndrome.  JAMA Neurol 73, 1009–1016. https://doi​.org/10.1001/jamaneurol​.2016.0449 [PMC free article: PMC5426359] [PubMed: 27367160]
37. Kanter-Schlifke, Irene, Georgievska, B., Kirik, D., Kokaia, M., 2007. Seizure suppression by GDNF gene therapy in animal models of epilepsy.  Mol Ther 15, 1106–1113. https://doi​.org/10.1038/sj.mt.6300148 [PubMed: 17387333]
38. Kanter-Schlifke, I., Toft Sørensen, A., Ledri, M., Kuteeva, E., Hökfelt, T., Kokaia, M., 2007. Galanin gene transfer curtails generalized seizures in kindled rats without altering hippocampal synaptic plasticity.  Neuroscience 150, 984–992. https://doi​.org/10.1016/j​.neuroscience.2007.09.056 [PubMed: 17988802]
39. Kätzel, D., Nicholson, E., Schorge, S., Walker, M.C., Kullmann, D.M., 2014. Chemical-genetic attenuation of focal neocortical seizures.  Nat Commun 5, 3847. https://doi​.org/10.1038/ncomms4847 [PMC free article: PMC4050272] [PubMed: 24866701]
40. Kovac, S., Walker, M.C., 2013. Neuropeptides in epilepsy.  Neuropeptides, Neuropeptides in Mental Health and Behaviour 47, 467–475. https://doi​.org/10.1016/j​.npep.2013.10.015 [PubMed: 24210141]
41. Krook-Magnuson, E., Armstrong, C., Oijala, M., Soltesz, I., 2013. On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy.  Nat Commun 4, 1376. https://doi​.org/10.1038/ncomms2376 [PMC free article: PMC3562457] [PubMed: 23340416]
42. Krook-Magnuson, E., Szabo, G.G., Armstrong, C., Oijala, M., Soltesz, I., 2014. Cerebellar Directed Optogenetic Intervention Inhibits Spontaneous Hippocampal Seizures in a Mouse Model of Temporal Lobe Epilepsy.  eNeuro 1, e.2014. https://doi​.org/10.1523/ENEURO​.0005-14.2014 [PMC free article: PMC4293636] [PubMed: 25599088]
43. Lang, M., Wither, R.G., Colic, S., Wu, C., Monnier, P.P., Bardakjian, B.L., Zhang, L., Eubanks, J.H., 2014. Rescue of behavioral and EEG deficits in male and female Mecp2-deficient mice by delayed Mecp2 gene reactivation.  Human Molecular Genetics 23, 303–318. https://doi​.org/10.1093/hmg/ddt421 [PMC free article: PMC3869352] [PubMed: 24009314]
44. Lenk, G.M., Jafar-Nejad, P., Hill, S.F., Huffman, L.D., Smolen, C.E., Wagnon, J.L., Petit, H., Yu, W., Ziobro, J., Bhatia, K., Parent, J., Giger, R.J., Rigo, F., Meisler, M.H., 2020. Scn8a Antisense Oligonucleotide Is Protective in Mouse Models of SCN8A Encephalopathy and Dravet Syndrome.  Annals of Neurology 87, 339–346. https://doi​.org/10.1002/ana.25676 [PMC free article: PMC7064908] [PubMed: 31943325]
45. Li, C., Samulski, R.J., 2020. Engineering adeno-associated virus vectors for gene therapy.  Nat Rev Genet 21, 255–272. https://doi​.org/10.1038​/s41576-019-0205-4 [PubMed: 32042148]
46. Lieb, A., Qiu, Y., Dixon, C.L., Heller, J.P., Walker, M.C., Schorge, S., Kullmann, D.M., 2018. Biochemical autoregulatory gene therapy for focal epilepsy.  Nat. Med. 24, 1324–1329. https://doi​.org/10.1038​/s41591-018-0103-x [PMC free article: PMC6152911] [PubMed: 29988123]
47. Lieb, A., Weston, M., Kullmann, D.M., 2019. Designer receptor technology for the treatment of epilepsy.  EBioMedicine 43, 641–649. https://doi​.org/10.1016/j​.ebiom.2019.04.059 [PMC free article: PMC6558262] [PubMed: 31078519]
48. Lin, E.-J.D., Richichi, C., Young, D., Baer, K., Vezzani, A., During, M.J., 2003. Recombinant AAV-mediated expression of galanin in rat hippocampus suppresses seizure development.  European Journal of Neuroscience 18, 2087–2092. https://doi​.org/10.1046/j​.1460-9568.2003.02926.x [PubMed: 14622242]
49. Lonser, R.R., Akhter, A.S., Zabek, M., Elder, J.B., Bankiewicz, K.S., 2020. Direct convective delivery of adeno-associated virus gene therapy for treatment of neurological disorders. J Neurosurg 1–13. https://doi​.org/10.3171/2020.4.JNS20701 [PubMed: 32915526]
50. Magloire, V., Cornford, J., Lieb, A., Kullmann, D.M., Pavlov, I., 2019. KCC2 overexpression prevents the paradoxical seizure-promoting action of somatic inhibition. Nat Commun 10, 1225. https://doi​.org/10.1038​/s41467-019-08933-4 [PMC free article: PMC6420604] [PubMed: 30874549]
51. Magloire, V., Mercier, M.S., Kullmann, D.M., Pavlov, I., 2018. GABAergic Interneurons in Seizures: Investigating Causality With Optogenetics. Neuroscientist 1073858418805002. https://doi​.org/10.1177/1073858418805002 [PMC free article: PMC6745605] [PubMed: 30317911]
52. Manvich, D.F., Webster, K.A., Foster, S.L., Farrell, M.S., Ritchie, J.C., Porter, J.H., Weinshenker, D., 2018. The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice.  Sci Rep 8, 3840. https://doi​.org/10.1038​/s41598-018-22116-z [PMC free article: PMC5832819] [PubMed: 29497149]
53. Mayford, M., Baranes, D., Podsypanina, K., Kandel, E.R., 1996. The 3′-untranslated region of CaMKIIα is a cis-acting signal for the localization and translation of mRNA in dendrites. PNAS 93, 13250–13255. https://doi​.org/10.1073/pnas.93.23.13250 [PMC free article: PMC24079] [PubMed: 8917577]
54. McCown, T.J., 2006. Adeno-associated Virus-Mediated Expression and Constitutive Secretion of Galanin Suppresses Limbic Seizure Activity in Vivo.  Molecular Therapy 14, 63–68. https://doi​.org/10.1016/j​.ymthe.2006.04.004 [PubMed: 16730475]
55. Melin, E., Nanobashvili, A., Avdic, U., Gøtzsche, C.R., Andersson, M., Woldbye, D.P.D., Kokaia, M., 2019. Disease Modification by Combinatorial Single Vector Gene Therapy: A Preclinical Translational Study in Epilepsy.  Molecular Therapy—Methods & Clinical Development 15, 179–193. https://doi​.org/10.1016/j​.omtm.2019.09.004 [PMC free article: PMC6807261] [PubMed: 31660420]
56. Mendell, J.R., Al-Zaidy, S., Shell, R., Arnold, W.D., Rodino-Klapac, L.R., Prior, T.W., Lowes, L., Alfano, L., Berry, K., Church, K., Kissel, J.T., Nagendran, S., L’Italien, J., Sproule, D.M., Wells, C., Cardenas, J.A., Heitzer, M.D., Kaspar, A., Corcoran, S., Braun, L., Likhite, S., Miranda, C., Meyer, K., Foust, K.D., Burghes, A.H.M., Kaspar, B.K., 2017. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy.  N. Engl. J. Med. 377, 1713–1722. https://doi​.org/10.1056/NEJMoa1706198 [PubMed: 29091557]
57. Morrell, M.J., RNS System in Epilepsy Study Group, 2011. Responsive cortical stimulation for the treatment of medically intractable partial epilepsy.  Neurology 77, 1295–1304. https://doi​.org/10.1212/WNL​.0b013e3182302056 [PubMed: 21917777]
58. Morris, G., O’Brien, D., Henshall, D.C., 2021. Opportunities and challenges for microRNA-targeting therapeutics for epilepsy.  Trends Pharmacol Sci 42, 605–616. https://doi​.org/10.1016/j​.tips.2021.04.007 [PubMed: 33992468]
59. Nagai, Y., Kikuchi, E., Lerchner, W., Inoue, K., Ji, B., Eldridge, M.A.G., Kaneko, H., Kimura, Y., Oh-Nishi, A., Hori, Y., Kato, Y., Hirabayashi, T., Fujimoto, A., Kumata, K., Zhang, M.-R., Aoki, I., Suhara, T., Higuchi, M., Takada, M., Richmond, B.J., Minamimoto, T., 2016. PET imaging-guided chemogenetic silencing reveals a critical role of primate rostromedial caudate in reward evaluation.  Nat Commun 7, 13605. https://doi​.org/10.1038/ncomms13605 [PMC free article: PMC5150653] [PubMed: 27922009]
60. Naso, M.F., Tomkowicz, B., Perry, W.L., Strohl, W.R., 2017. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy.  BioDrugs 31, 317–334. https://doi​.org/10.1007​/s40259-017-0234-5 [PMC free article: PMC5548848] [PubMed: 28669112]
61. Natarajan, G., Leibowitz, J.A., Zhou, J., Zhao, Y., McElroy, J.A., King, M.A., Ormerod, B.K., Carney, P.R., 2017. Adeno-associated viral vector-mediated preprosomatostatin expression suppresses induced seizures in kindled rats.  Epilepsy Research 130, 81–92. https://doi​.org/10.1016/j​.eplepsyres.2017.01.002 [PubMed: 28167431]
62. Niibori, Y., Lee, S.J., Minassian, B.A., Hampson, D.R., 2020. Sexually Divergent Mortality and Partial Phenotypic Rescue After Gene Therapy in a Mouse Model of Dravet Syndrome.  Hum Gene Ther 31, 339–351. https://doi​.org/10.1089/hum.2019.225 [PMC free article: PMC7087406] [PubMed: 31830809]
63. Noè, F., Pool, A.-H., Nissinen, J., Gobbi, M., Bland, R., Rizzi, M., Balducci, C., Ferraguti, F., Sperk, G., During, M.J., Pitkänen, A., Vezzani, A., 2008. Neuropeptide Y gene therapy decreases chronic spontaneous seizures in a rat model of temporal lobe epilepsy.  Brain 131, 1506–1515. https://doi​.org/10.1093/brain/awn079 [PubMed: 18477594]
64. Noe, F., Vaghi, V., Balducci, C., Fitzsimons, H., Bland, R., Zardoni, D., Sperk, G., Carli, M., During, M.J., Vezzani, A., 2010. Anticonvulsant effects and behavioural outcomes of rAAV serotype 1 vector-mediated neuropeptide Y overexpression in rat hippocampus.  Gene Ther 17, 643–652. https://doi​.org/10.1038/gt.2010.23 [PubMed: 20220782]
65. Paradiso, B., Marconi, P., Zucchini, S., Berto, E., Binaschi, A., Bozac, A., Buzzi, A., Mazzuferi, M., Magri, E., Mora, G.N., Rodi, D., Su, T., Volpi, I., Zanetti, L., Marzola, A., Manservigi, R., Fabene, P.F., Simonato, M., 2009. Localized delivery of fibroblast growth factor–2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model.  PNAS 106, 7191–7196. https://doi​.org/10.1073/pnas.0810710106 [PMC free article: PMC2678472] [PubMed: 19366663]
66. Paradiso, B., Zucchini, S., Su, T., Bovolenta, R., Berto, E., Marconi, P., Marzola, A., Navarro Mora, G., Fabene, P.F., Simonato, M., 2011. Localized overexpression of FGF-2 and BDNF in hippocampus reduces mossy fiber sprouting and spontaneous seizures up to 4 weeks after pilocarpine-induced status epilepticus.  Epilepsia 52, 572–578. https://doi​.org/10.1111/j​.1528-1167.2010.02930.x [PubMed: 21269288]
67. Pausch, P., Al-Shayeb, B., Bisom-Rapp, E., Tsuchida, C.A., Li, Z., Cress, B.F., Knott, G.J., Jacobsen, S.E., Banfield, J.F., Doudna, J.A., 2020. CRISPR-CasΦ from huge phages is a hypercompact genome editor. Science 369, 333–337. https://doi​.org/10.1126/science.abb1400 [PMC free article: PMC8207990] [PubMed: 32675376]
68. Paz, J.T., Davidson, T.J., Frechette, E.S., Delord, B., Parada, I., Peng, K., Deisseroth, K., Huguenard, J.R., 2013. Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury.  Nat. Neurosci. 16, 64–70. https://doi​.org/10.1038/nn.3269 [PMC free article: PMC3700812] [PubMed: 23143518]
69. Powell, K.L., Fitzgerald, X., Shallue, C., Jovanovska, V., Klugmann, M., Von Jonquieres, G., O’Brien, T.J., Morris, M.J., 2018. Gene therapy mediated seizure suppression in Genetic Generalised Epilepsy: Neuropeptide Y overexpression in a rat model.  Neurobiol Dis 113, 23–32. https://doi​.org/10.1016/j​.nbd.2018.01.016 [PubMed: 29414380]
70. Raol, Y.H., Lund, I.V., Bandyopadhyay, S., Zhang, G., Roberts, D.S., Wolfe, J.H., Russek, S.J., Brooks-Kayal, A.R., 2006. Enhancing GABAA Receptor α1 Subunit Levels in Hippocampal Dentate Gyrus Inhibits Epilepsy Development in an Animal Model of Temporal Lobe Epilepsy. J. Neurosci. 26, 11342–11346. https://doi​.org/10.1523/JNEUROSCI​.3329-06.2006 [PMC free article: PMC6674546] [PubMed: 17079662]
71. Ravindra Kumar, S., Miles, T.F., Chen, X., Brown, D., Dobreva, T., Huang, Q., Ding, X., Luo, Y., Einarsson, P.H., Greenbaum, A., Jang, M.J., Deverman, B.E., Gradinaru, V., 2020. Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.  Nat Methods 17, 541–550. https://doi​.org/10.1038​/s41592-020-0799-7 [PMC free article: PMC7219404] [PubMed: 32313222]
72. Richichi, C., Lin, E.-J.D., Stefanin, D., Colella, D., Ravizza, T., Grignaschi, G., Veglianese, P., Sperk, G., During, M.J., Vezzani, A., 2004. Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus.  J Neurosci 24, 3051–9. https://doi​.org/10.1523/JNEUROSCI​.4056-03.2004 [PMC free article: PMC6729841] [PubMed: 15044544]
73. Ruffmann, C., Bogliun, G., Beghi, E., 2006. Epileptogenic drugs: a systematic review.  Expert Rev Neurother 6, 575–589. https://doi​.org/10.1586/14737175.6.4.575 [PubMed: 16623656]
74. Sahel, J.-A., Boulanger-Scemama, E., Pagot, C., Arleo, A., Galluppi, F., Martel, J.N., Esposti, S.D., Delaux, A., de Saint Aubert, J.-B., de Montleau, C., Gutman, E., Audo, I., Duebel, J., Picaud, S., Dalkara, D., Blouin, L., Taiel, M., Roska, B., 2021. Partial recovery of visual function in a blind patient after optogenetic therapy.  Nat Med 27, 1223–1229. https://doi​.org/10.1038​/s41591-021-01351-4 [PubMed: 34031601]
75. Sinnett, S.E., Boyle, E., Lyons, C., Gray, S.J., 2021. Engineered microRNA-based regulatory element permits safe high-dose miniMECP2 gene therapy in Rett mice. Brain. https://doi​.org/10.1093/brain/awab182 [PMC free article: PMC8783608] [PubMed: 33950254]
76. Snowball, A., Chabrol, E., Wykes, R.C., Shekh-Ahmad, T., Cornford, J.H., Lieb, A., Hughes, M.P., Massaro, G., Rahim, A.A., Hashemi, K.S., Kullmann, D.M., Walker, M.C., Schorge, S., 2019. Epilepsy Gene Therapy Using an Engineered Potassium Channel.  J. Neurosci. 39, 3159–3169. https://doi​.org/10.1523/JNEUROSCI​.1143-18.2019 [PMC free article: PMC6468110] [PubMed: 30755487]
77. Sørensen, A.T., Nikitidou, L., Ledri, M., Lin, E.-J.D., During, M.J., Kanter-Schlifke, I., Kokaia, M., 2009. Hippocampal NPY gene transfer attenuates seizures without affecting epilepsy-induced impairment of LTP.  Exp Neurol 215, 328–333. https://doi​.org/10.1016/j​.expneurol.2008.10.015 [PMC free article: PMC2896682] [PubMed: 19038255]
78. Suzuki, K., Tsunekawa, Y., Hernandez-Benitez, R., Wu, J., Zhu, J., Kim, E.J., Hatanaka, F., Yamamoto, M., Araoka, T., Li, Z., Kurita, M., Hishida, T., Li, M., Aizawa, E., Guo, S., Chen, S., Goebl, A., Soligalla, R.D., Qu, J., Jiang, T., Fu, X., Jafari, M., Esteban, C.R., Berggren, W.T., Lajara, J., Nuñez-Delicado, E., Guillen, P., Campistol, J.M., Matsuzaki, F., Liu, G.-H., Magistretti, P., Zhang, Kun, Callaway, E.M., Zhang, Kang, Belmonte, J.C.I., 2016. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.  Nature 540, 144–149. https://doi​.org/10.1038/nature20565 [PMC free article: PMC5331785] [PubMed: 27851729]
79. Takeuchi, Y., Harangozó, M., Pedraza, L., Földi, T., Kozák, G., Li, Q., Berényi, A., 2021. Closed-loop stimulation of the medial septum terminates epileptic seizures.  Brain 144, 885–908. https://doi​.org/10.1093/brain/awaa450 [PubMed: 33501929]
80. The MECP2 duplication syndrome—Ramocki—2010—American Journal of Medical Genetics Part A—Wiley Online Library [WWW Document], n.d. URL https://onlinelibrary.wiley.com/doi/10.1002/ajmg.a.33184 (accessed 8.5.21). [PMC free article: PMC2861792] [PubMed: 20425814]
81. Turner, T.J., Zourray, C., Schorge, S., Lignani, G., 2021. Recent advances in gene therapy for neurodevelopmental disorders with epilepsy.  Journal of Neurochemistry 157, 229–262. https://doi​.org/10.1111/jnc.15168 [PMC free article: PMC8436749] [PubMed: 32880951]
82. University College, London, 2020. Phase I/IIa, First-in-human, Open-label, Single-site Trial of In-vivo Lentiviral Engineered Potassium (K+) Channel (EKC) Gene Therapy for Refractory Epilepsy (Clinical trial registration No. NCT04601974). clinicaltrials.gov.
83. Vormstein-Schneider, D., Lin, J.D., Pelkey, K.A., Chittajallu, R., Guo, B., Arias-Garcia, M.A., Allaway, K., Sakopoulos, S., Schneider, G., Stevenson, O., Vergara, J., Sharma, J., Zhang, Q., Franken, T.P., Smith, J., Ibrahim, L.A., Mastro, K.J., Sabri, E., Huang, S., Favuzzi, E., Burbridge, T., Xu, Q., Guo, L., Vogel, I., Sanchez, V., Saldi, G.A., Gorissen, B.L., Yuan, X., Zaghloul, K.A., Devinsky, O., Sabatini, B.L., Batista-Brito, R., Reynolds, J., Feng, G., Fu, Z., McBain, C.J., Fishell, G., Dimidschstein, J., 2020. Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans.  Nat Neurosci 23, 1629–1636. https://doi​.org/10.1038​/s41593-020-0692-9 [PMC free article: PMC8015416] [PubMed: 32807948]
84. Walker, M.C., Kullmann, D.M., 2020. Optogenetic and chemogenetic therapies for epilepsy.  Neuropharmacology 168, 107751. https://doi​.org/10.1016/j​.neuropharm.2019.107751 [PubMed: 31494141]
85. Wallace, R.H., Wang, D.W., Singh, R., Scheffer, I.E., George, A.L., Phillips, H.A., Saar, K., Reis, A., Johnson, E.W., Sutherland, G.R., Berkovic, S.F., Mulley, J.C., 1998. Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta1 subunit gene SCN1B.  Nat. Genet 19, 366–370. https://doi​.org/10.1038/1252 [PubMed: 9697698]
86. Wang, Ying, Liang, J., Chen, L., Shen, Y., Zhao, J., Xu, C., Wu, X., Cheng, H., Ying, X., Guo, Y., Wang, S., Zhou, Y., Wang, Yi, Chen, Z., 2018. Pharmaco-genetic therapeutics targeting parvalbumin neurons attenuate temporal lobe epilepsy.  Neurobiol. Dis. 117, 149–160. https://doi​.org/10.1016/j​.nbd.2018.06.006 [PubMed: 29894753]
87. Wanisch, K., Yáñez-Muñoz, R.J., 2009. Integration-deficient lentiviral vectors: a slow coming of age.  Mol Ther 17, 1316–1332. https://doi​.org/10.1038/mt.2009.122 [PMC free article: PMC2835240] [PubMed: 19491821]
88. Weston, M., Kaserer, T., Wu, A., Mouravlev, A., Carpenter, J.C., Snowball, A., Knauss, S., von Schimmelmann, M., During, M.J., Lignani, G., Schorge, S., Young, D., Kullmann, D.M., Lieb, A., 2019. Olanzapine: A potent agonist at the hM4D(Gi) DREADD amenable to clinical translation of chemogenetics.  Sci Adv 5, eaaw1567. https://doi​.org/10.1126/sciadv.aaw1567 [PMC free article: PMC6469940] [PubMed: 31001591]
89. Wicker, E., Forcelli, P.A., 2021. Optogenetic activation of the reticular nucleus of the thalamus attenuates limbic seizures via inhibition of the midline thalamus. Epilepsia. https://doi​.org/10.1111/epi.17016 [PMC free article: PMC9092275] [PubMed: 34309008]
90. Wicker, E., Forcelli, P.A., 2016. Chemogenetic silencing of the midline and intralaminar thalamus blocks amygdala-kindled seizures.  Exp. Neurol. 283, 404–412. https://doi​.org/10.1016/j​.expneurol.2016.07.003 [PMC free article: PMC4992629] [PubMed: 27404844]
91. Woldbye, D.P.D., Angehagen, M., Gøtzsche, C.R., Elbrønd-Bek, H., Sørensen, A.T., Christiansen, S.H., Olesen, M.V., Nikitidou, L., Hansen, T.V.O., Kanter-Schlifke, I., Kokaia, M., 2010. Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures.  Brain 133, 2778–2788. https://doi​.org/10.1093/brain/awq219 [PubMed: 20688813]
92. Wykes, R.C., Heeroma, J.H., Mantoan, L., Zheng, K., MacDonald, D.C., Deisseroth, K., Hashemi, K.S., Walker, M.C., Schorge, S., Kullmann, D.M., 2012. Optogenetic and potassium channel gene therapy in a rodent model of focal neocortical epilepsy.  Sci Transl Med 4, 161ra152. https://doi​.org/10.1126/scitranslmed​.3004190 [PMC free article: PMC3605784] [PubMed: 23147003]
93. Xiong, W., Wu, D.M., Xue, Y., Wang, S.K., Chung, M.J., Ji, X., Rana, P., Zhao, S.R., Mai, S., Cepko, C.L., 2019. AAV cis-regulatory sequences are correlated with ocular toxicity.  Proc Natl Acad Sci U S A 116, 5785–5794. https://doi​.org/10.1073/pnas.1821000116 [PMC free article: PMC6431174] [PubMed: 30833387]
94. Yamagata, T., Raveau, M., Kobayashi, K., Miyamoto, H., Tatsukawa, T., Ogiwara, I., Itohara, S., Hensch, T.K., Yamakawa, K., 2020. CRISPR/dCas9-based Scn1a gene activation in inhibitory neurons ameliorates epileptic and behavioral phenotypes of Dravet syndrome model mice.  Neurobiology of Disease 141, 104954. https://doi​.org/10.1016/j​.nbd.2020.104954 [PubMed: 32445790]
95. Young, A., Tanenhaus, A., Chen, M., et al, 2019. A GABA-Selective AAV Vector-Based Approach to Up-Regulate Endogenous Scn1a Expression Reverses Key Phenotypes in a Mouse Model of Dravet Syndrome. ASGCT Abstracts.
96. Yu, F.H., Mantegazza, M., Westenbroek, R.E., Robbins, C.A., Kalume, F., Burton, K.A., Spain, W.J., McKnight, G.S., Scheuer, T., Catterall, W.A., 2006. Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy.  Nat. Neurosci 9, 1142–1149. https://doi​.org/10.1038/nn1754 [PubMed: 16921370]
97. Zafar, R., King, M.A., Carney, P.R., 2012. Adeno associated viral vector-mediated expression of somatostatin in rat hippocampus suppresses seizure development.  Neurosci Lett 509, 87–91. https://doi​.org/10.1016/j​.neulet.2011.12.035 [PubMed: 22245439]
98. Zufferey, R., Dull, T., Mandel, R.J., Bukovsky, A., Quiroz, D., Naldini, L., Trono, D., 1998. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.  J Virol 72, 9873–9880. https://doi​.org/10.1128/JVI​.72.12.9873-9880.1998 [PMC free article: PMC110499] [PubMed: 9811723]